Our long term objective is to understand how chromosomal segregation takes place in bacteria, i.e., what is the equivalent of mitosis in prokaryotes? We believe that such knowledge will be useful for a general understanding of cell function and, in particular, to help define new targets for antibacterial chemotherapy. This proposal is based on our observation that the replication origin of the Escherichia coli chromosome binds to the membrane for a defined period of time during the cell cycle. We have postulated that this time represent a biological clock during which the incipient progeny chromosomes become relegated to the two cell halves. During this period, origin DNA is hemimethylated, that is, it is newly replicated, but has not yet been modified by the major E. coli methylase, Dam. We have found a membrane protein we call Hob that binds uniquely to hemimethylated origin DNA. We have also delineated the gene for this protein, hob, to within a lambdal phage of the Kohara collection. The E. coli DNA inserted into this phage includes a gene, pcsA, involved in chromosome segregation. Cold sensitive mutants in pcsA cannot partition their chromosome and are defective in cell division. We wish to determine whether the Hob protein acts to anchor the replicative origin to the membrane (thus functioning as part of a bacterial kinetochore-equivalent). Because this is a novel topic, we must first carry out considerable biochemical and genetic groundwork. To this end, we will ask the following questions: What is the sequence of Hob? Are the hob and pcsA genes the same? Is hob essential? What are cytological features of hob mutants? We will then study the sequence in oriC that is recognized by Hob, the specificity of the interaction, and the state of aggregation of Hob in solution. Because the Hob protein is unlikely to function in isolation, we will determine what other proteins it interacts with by looking for extragenic suppressors of conditional hob mutations. We will determine the intracellular localization of the Hob protein and possible "suppressor" proteins. Our model makes a strong prediction about the time in the cell cycle when the Hob protein binds to the DNA. We will test this notion by in vivo footprinting techniques using cells synchronized in their DNA replication. This study will also reveal the DNA "occupancy time" of other proteins during the initiation and early replication of the E. coli chromosome. Ultimately, the function of the Hob protein must be studied by physiological means. We recognize that such studies are difficult and must rely on a firm biochemical and genetic knowledge of the system. Should we make sufficient progress during the period of this grant, we will study the activity and regulation of Hob during the cell cycle, in cultures growing at different rates, and in the presence of different amounts of oriC in the cell.